Rotavirus_Barros_etal_2018-PlosOne

Prévia do material em texto

RESEARCH ARTICLE
Rotavirus A in wild and domestic animals from
areas with environmental degradation in the
Brazilian Amazon
Bruno de Cássio Veloso de BarrosID
1, Elaine Nunes ChagasID
1, Luna Wanessa Bezerra2,
Laila Graziela Ribeiro2, Jose Wandilson Barboza Duarte JúniorID
2, Diego Pereira2,
Edvaldo Tavares da Penha Junior1, Julia Rezende Silva3, Delana Andreza Melo Bezerra1,
Renato Silva Bandeira1, Helder Henrique Costa PinheiroID
4, Sylvia de Fátima dos
Santos Guerra1,2, Ricardo José de Paula Souza e Guimarães1, Joana D’Arc
Pereira Mascarenhas1*
1 Evandro Chagas Institute, Ministry of Health, Ananindeua, Pará, Brazil, 2 Amazon Metropolitan University
Center, Belém, Pará, Brazil, 3 University of the State of Pará, Belém, Pará, Brazil, 4 Federal University of
Pará, Belém, Pará, Brazil
* joanamascarenhas@iec.gov.br
Abstract
Acute gastroenteritis is one of the main causes of mortality in humans and young animals.
Domestic and mainly wild animals such as bats, small rodents and birds are highly diversi-
fied animals in relation to their habitats and ecological niches and are widely distributed geo-
graphically in environments of forest fragmentation in some areas of the Amazon, being
considered important sources for viruses that affect humans and other animals. Due to the
anthropical activities, these animals changed their natural habitat and adapted to urbanized
environments, thus representing risks to human and animal health. Although the knowledge
of the global diversity of enteric viruses is scarce, there are reports demonstrating the detec-
tion of rotavirus in domestic animals and animals of productive systems, such as bovines
and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples
of different animal species from the northeastern mesoregion of the state of Pará, Brazil,
which is characterized as an urbanized area with forest fragments. The fecal specimens
were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time
Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed
that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples
distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%),
horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating
the circulation of RVA in domestic animals and suggesting that such proximity could cause
transmissions between different species and the occurrence of rearrangements in the
genome of RVA as already described in the literature, associated to the traces of environ-
mental degradation in the studied areas.
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 1 / 18
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Barros BdCVd, Chagas EN, Bezerra LW,
Ribeiro LG, Duarte Júnior JWB, Pereira D, et al.
(2018) Rotavirus A in wild and domestic animals
from areas with environmental degradation in the
Brazilian Amazon. PLoS ONE 13(12): e0209005.
https://doi.org/10.1371/journal.pone.0209005
Editor: Dong-Yan Jin, University of Hong Kong,
HONG KONG
Received: April 27, 2018
Accepted: November 28, 2018
Published: December 18, 2018
Copyright: © 2018 Barros et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript.
Funding: This research was supported by the
Coordination for the Improvement of Higher
Education Personnel (CAPES) n. 2303800717 /
2013-52, National Council for Scientific and
Technological Development (CNPq) and Evandro
Chagas Institute, Ministry of Health, Ananindeua,
Brazil. BARROS BCV is a beneficiary of the Capes
grant; Chagas EHN and Mascarenhas JDP are
recipients of grants from CNPq. The authors would
http://orcid.org/0000-0002-1241-8258
http://orcid.org/0000-0002-3623-1711
http://orcid.org/0000-0002-4801-2356
http://orcid.org/0000-0001-5567-3550
https://doi.org/10.1371/journal.pone.0209005
http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0209005&domain=pdf&date_stamp=2018-12-18
http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0209005&domain=pdf&date_stamp=2018-12-18
http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0209005&domain=pdf&date_stamp=2018-12-18
http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0209005&domain=pdf&date_stamp=2018-12-18
http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0209005&domain=pdf&date_stamp=2018-12-18
http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0209005&domain=pdf&date_stamp=2018-12-18
https://doi.org/10.1371/journal.pone.0209005
http://creativecommons.org/licenses/by/4.0/
Introduction
Emerging and reemerging infectious diseases are increasing each year in several countries,
with an impact both on human populations and on domestic and wild animals living in areas
with considerable forest remnants [1]. Most of these diseases are of viral origin, suggesting the
emergence and reemergence of viruses that are triggered by human activities that modify the
environment [2].
The populations of wild animals that inhabit forest fragments are strategic groups for stud-
ies of public health and the transmission of zoonosis, given that they act as indicators in the
assistance and intervention in the human populations, aiming at the prevention of outbreaks
and epidemics [3].
Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by dif-
ferent infectious or parasite agents [4–7]. They represent one of the main causes of mortality
in humans, and in young animals, counting for about 25% of mortality [8]. Rotavirus is widely
distributed in animals, which act as sources of rotavirus emergent strains, with these animals
acting in the transmission between species and through reassortment leading to the emergence
of new strains which have been reported in human infections [9–12].
The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as
Rotavirus group A to I, with a recent proposal of the J species [13, 14]. Rotavirus A (RVA) is
widespread worldwide and predominantly infects humans, bovines and other mammal spe-
cies, as well as birds [15]. They have a double-stranded ribonucleic acid (dsRNA) genome,
divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and non-
structural (NSP1-NSP5/NSP6) proteins [16, 17].
There are records of a close relationship between Amazonian wildlife and human popula-
tions [18], and this interaction is the effect of anthropogenic urbanization activities that result
in the deforestation of forest areas, causing the degradation of previously isolated sites such as
caves and small caves, a continuous and nature progressive process that has led not only to
changes in wildlife habitats but also to a greater relationship with human populations in rural
and urban environments, contributing to the occurrence and emergence of diseases different
from what normally occurs in endemic regions [19–22].
Although the results of RVA have already been described globally [12, 23–30], in Brazil, the
occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering
the large number of present species [4, 31–34].
In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast
metropolitan mesoregions are some of the areas with the highest indexes of environmental
changes [35], which are concentrated, along with the fact that the knowledge of the global
diversity of enteric virus in animals is scarce [36].
Therefore, it is important to monitor the health of domestic and wild animals in their natu-
ralhabitat, especially in areas with anthropic alterations that have an interface with rural com-
munities and enterprises, in order to investigate the occurrence of RVA in this population.
These communities are ecologically complex, because they have multiple hosts and endless
pathogens that may eventually circulate in contiguous urban centers, in addition to the fact
that it should also be considered that there is still a lack of studies showing the significance of
these viruses infecting this population, as in the context of epidemiological surveillance, these
animals become important, since they can be considered as natural sources, with the possibil-
ity of transmission to humans [37–39].
The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and
the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3
(NSP3), which was previously used in samples from human origin and with low viral loads
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 2 / 18
like to acknowledge the Amazon Support
Foundation of the studies and research (FAPESPA)
for funding (ICAAF166/2014). Thanks also to the
Evandro Chagas Institute for financial and
technicoscientific support for the execution of this
work. Finally, thanks are due to Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior
(CAPES) for awarding doctoral and pós doctoral’
scholarships. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
https://doi.org/10.1371/journal.pone.0209005
according to studies conducted by Zeng et al. [40] and Mijatovic et al. [41]. In the present
study, this assay was adopted as a screening test for the detection of RV in asymptomatic ani-
mal populations.
Therefore, the objective of the present study was to estimate the number of animals infected
by RVA in the forest environment and the possible environmental impacts, and the geographi-
cal distribution of this agent in the region.
Material and methods
Study area
The study area included the settlements of Expedito Ribeiro (Santa Bárbara do Pará), Vila
Ananin (Peixe-Boi) and Açaiteua-Centro Alegre (Viseu), located in areas with environmental
degradation in the state of Pará, in the Brazilian Amazon. According to Alvares et al. [42], the
city of Santa Bárbara do Pará presents a tropical type of motion-type Am and Viseu presents
atype of motion, with dry seasons in summer and winter-Am, As and Aw (Fig 1), taking into
account the weather forecast classification of Köppen-Geiger.
The collection points of the positive samples were georeferenced and the data were trans-
ported to the ArcGIS 10.3 program for the construction of distribution maps for the RVA at
the interface between agroecosystems and natural ecosystems.
Data extraction and geoprocessing analysis
Precipitation data were obtained from The Brazilian National Institute of Meteorology (Inmet -
http://www.inmet.gov.br/) for the years of capture in the Expedito Ribeiro Settlement (2014)
and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from
Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu.
Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field.
The municipal boundaries were obtained on the website of the Brazilian Institute of Geog-
raphy and Statistics (IBGE) (http://www.ibge.gov.br/) and data on deforestation and land use
were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data
in digital format since 2000 and TerraClass presents biannual data since 2004.
The satellite image was generated using the sensor Sentinel 2 of the European Space Agency
(ESA) (https://sentinel.esa.int/ web/sentinel/user-guides/sentinel-2-msi) with Open Access
CC-BY License (http://open.esa.int/) from the years of 2017 and 2018.
All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/
stored in a GIS for the editing of the graphic elements, establishment of topological relations
between the elements and their respective attributes, spatial analysis and visualization of the
result through thematic maps.
Choice of study areas
For the present study, forest fragments of similar size, shape and Phyto physiology were cho-
sen, considering an open peri urban matrix with similar soil use. The selected fragments were
distributed within the mesoregions studied, and in each selected fragment fecal samples were
randomly collected from domestic and wild animals [45].
Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because
the study sites were in an area with a high cloud presence, which prevented observation (the
area was not observed).
The data processing, interpretation, visualization and spatial analysis were performed in
ArcGIS software (http://www.arcgis.com/).
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 3 / 18
http://www.inmet.gov.br/
http://www.ibge.gov.br/
https://sentinel.esa.int/
http://open.esa.int/
http://www.arcgis.com/
https://doi.org/10.1371/journal.pone.0209005
For the analysis of data related to the determination of the richness, composition and abun-
dance of the fauna of the animals studied in the study area, considering the collection methods
adopted and the species available in each city, each sample was considered as an independent
sample.
Statistical analysis
The richness of wild fauna and domestic animals was determined by the total number of spe-
cies including all collection methods, and the similarity of species was made by the chi-square
analysis between the samples of the different treatments with the aid of the EstimateS 8.0 soft-
ware [46].
For the calculation of the Test T, the Statistica software was used, and the indices of infected
animals in the two environments (forest fragment and peridomicile) were calculated for each
treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the
values of the diversity indexes through the paired test, as well as the descriptive analysis of the
anthropic effects [47].
Fig 1. Place of study and capture of the animals.
https://doi.org/10.1371/journal.pone.0209005.g001
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 4 / 18
https://doi.org/10.1371/journal.pone.0209005.g001
https://doi.org/10.1371/journal.pone.0209005
The data obtained for the occurrence of RVA and the questionnaires was inserted into a
database for a descriptive analysis of the epidemiological profile of the animal population in
the three forest ecosystems studied. In this analysis, descriptive statistical treatments were car-
ried out, using customized "row-columns" type charts, referring to the data, in order to charac-
terize the sample and quantify the results using absolute frequency values using the chi-square
test and the Test T.
Ethical aspects
Population study, collection of clinical specimens and laboratory methodology. The
flying animals (wild birds and chiroptera) were captured using mist nets which were opened at
dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour
until the closing, with a sampling effort of 15 days. This research was approved by National
Council for Animal Control and Experimentation (CONCEA), System of Authorization and
Information in Biodiversity—SISBIO/ICMBIO/Ministry of the Environment under No.
37174–1 and by Committee on Ethics in Animal Use (CEUA / IEC) No. 019/2014.
All procedures with animals were performed by veterinarians, being birds and bats identi-
fied and released at the same capturesite. The fecal specimens were collected by stimulation of
the rectal ampulla with the use of a "Zaragatoa", packed in cryogenic vials, identified, stored in
liquid nitrogen, and later sent to the Laboratory.
Wild animals (small non-flying mammals) were trapped within live-containment traps of the
Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sam-
ple plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture
made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple
and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged
when necessary and later after the capture in bags of cloth and at least five specimens of each spe-
cies were chosen for the collection of biological material. The wild animals were sedated with a
combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, eutha-
nized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recom-
mendation of the National Council for the Control of Animal Experimentation (CONCEA).
From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domes-
tic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and
handled in Level Three Biosafety Laboratory (NB3).
The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-
GEN, USA/KIT QIAGEN), following the manufacturer’s recommendations, with minor adap-
tation according to the protocol described in the supplemental data.
The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using
the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture
containing: RNAse-free H2O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x),
primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM),
probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and
reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing
of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute.
The analyzes were considered positive when presenting the cycle threshold (CT)� 40. In
order to guarantee a reliable test result, the measurements of contamination control were per-
formed with the use of positive animal control (SA11 prototype) and a negative control (ultra-
pure water).
All RVA-positive samples were subjected to reverse transcription-polymerase chain reac-
tion (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 5 / 18
https://doi.org/10.1371/journal.pone.0209005
round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was
conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified
and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were col-
lected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The
sequence fragments were assembled and edited using the Geneious Bioinformatics software
platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National
Center for Biotechnology Information GenBank database using BLAST alignment tool to elu-
cidate the RVA genotype of the samples.
Results
From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals
belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR,
and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines
(21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%)
and felines (3.37%). The CT interval ranged from 28.47 to 39.9 (mean of 36.79) with the fol-
lowing mean values per species: horses (34.11), birds (34.24), cattle (36.02), canines and chi-
ropterans (37.1), rodents (37.76), swine (37.91) and felines (38.36).
It was possible to detect viral strains in all genders of animals studied and in the harvesting
period none of the animals showed signs of acute infection and / or diarrhea.
Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to
the three areas of forest fragment, according to Fig 2.
In relation to the evaluated bovines, only in the city of Viseu, these species were studied
because they were created extensively. In addition, most of the animals were young with ages
varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance
and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low
weight performance and poor sanitary management status. In relation to chiroptera, 32
(17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with
12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5
(15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Arti-
beus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came
from areas of forest fragments located near bovine and equine farms, in addition to inhabiting
small chicken farms.
Fig 3 shows the results obtained for all the species of animals investigated in the forest frag-
ment as well as in the peridomicillus area.
The anthropic variables were analyzed for the three cities studied, as well as the use of the
soil within the range of the animals, obeying the domicile, the peridomicile and the forest frag-
ment where the traps of small rodents, birds and various species of animals were captured (Fig
4 and Fig 5).
Considering the factors related to the anthropic activities in the three studied areas within the
three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a
better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa
Bárbara, a greater concentration of occupations was observed around the area of forest fragment.
It was observed in this chosen area of the city, the presence of different families living in a rural
settlement, surviving from the exploitation of forest resources and the creation of small animals
for subsistence, such as poultry and fish farming, as well as family farming products.
The breeding of animals in native pastures was only observed in the cities of Peixe Boi and
Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small
animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 6 / 18
https://doi.org/10.1371/journal.pone.0209005
had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a
higher regeneration was observed in the pastures during the period of the study, with signifi-
cant secondary vegetation.
When comparing the climates of the three areas it was observed that the predominant cli-
mate is megathermal and humid with average annual temperature around 27˚C. The months
of October, November and December are the hottest, with temperatures between 32˚C and
34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around
2,350 mm, but strongly concentrated from January to June (80%). From September to Decem-
ber, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water defi-
cit in those months. The relative humidity of the average air oscillates around 85%, as shown
in Fig 6 [48].
The description of the accumulated precipitation in the year of capture of the fecal speci-
mens compared to the ClimatologicalNormals (CLINO) for the period from 1961–1990 of the
PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD),
Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rain-
fall in the regions, which facilitates the renewal of the pastures and the regeneration of the
impacted forests, being an important indicator of the reduction of the damages caused by
deforestation in the region.
Fig 2. Frequency of Rotavirus A in domestic and wild animals in the Brazilian Amazon from 2014 to 2016.
https://doi.org/10.1371/journal.pone.0209005.g002
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 7 / 18
https://doi.org/10.1371/journal.pone.0209005.g002
https://doi.org/10.1371/journal.pone.0209005
The average deforestation index in the three study areas was calculated from data obtained
from INPE information systems. It was observed that in the years of 2013 to 2014 there were
no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu
was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes
were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that
changes in the natural ecosystem may be associated with the frequencies for RVA in the stud-
ied areas, according to Fig 7.
When assessing the infected animals in relation to the uninfected animals in both the forest
fragment and the peridomicile, considering as animals of the forest fragment the birds, the
chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs,
felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12%
infected forest fragment animals (92/416) were obtained. Applying the selected statistical anal-
ysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p<0.001, meaning that
the hypothesis was corroborated, that is, the greater the degradation of the environment, the
more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the
Fig 3. Frequency of Rotavirus A in domestic and wild animals in the Brazilian Amazon from 2014 to 2016.
https://doi.org/10.1371/journal.pone.0209005.g003
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 8 / 18
https://doi.org/10.1371/journal.pone.0209005.g003
https://doi.org/10.1371/journal.pone.0209005
forest or even in the peri domiciliary region. In this sense, the possibility of contagion with
other species of animals, even humans, should be considered because of the capacity of the
rotavirus to be transmitted via the fecal / oral route or through direct contact with the environ-
ment. It is important to point out that the animals detected in this study are important sources
of viral strains.
A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4
gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P[6] genotype
and 6 to P[4]-type, according to Fig 8.
Fig 4. Map of distance of 3 km at the capture sites of the bats in (A) Santa Bárbara, (B) Viseu and (C) Peixe-Boi, with land use classes.
https://doi.org/10.1371/journal.pone.0209005.g004
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 9 / 18
https://doi.org/10.1371/journal.pone.0209005.g004
https://doi.org/10.1371/journal.pone.0209005
Discussion
In the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic
animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low
viral load of RVA in different species, which further correlates with the deforestation index.
These data are important because there is a lack of tests for RVA diagnosis in animals, since
the current methods of RVA detection does not always detect in these populations [8]. With
the advent of real-time PCR (qPCR), there was an exponential growth, compared to conven-
tional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is
Fig 5. Shows the percentage of each class of soil use within a 3 km radius of the place of capture of the animals in
each city studied.
https://doi.org/10.1371/journal.pone.0209005.g005
Fig 6. Accumulated precipitation of month X precipitation (CLINO 1961–1990) of the Belém (A) and Tracauateua
(B) PCDs.
https://doi.org/10.1371/journal.pone.0209005.g006
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 10 / 18
https://doi.org/10.1371/journal.pone.0209005.g005
https://doi.org/10.1371/journal.pone.0209005.g006
https://doi.org/10.1371/journal.pone.0209005
Fig 7. Positivity to RVA correlated with deforestation in the three cities of the Brazilian Amazon. Source: PRODES (http://www.dpi.inpe.br/prodesdigital/
prodesmunicipal.php).
https://doi.org/10.1371/journal.pone.0209005.g007
Fig 8. Results of nested and qRT-PCR for samples characterized in accordance to the host and localization.
https://doi.org/10.1371/journal.pone.0209005.g008
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 11 / 18
http://www.dpi.inpe.br/prodesdigital/prodesmunicipal.php
http://www.dpi.inpe.br/prodesdigital/prodesmunicipal.php
https://doi.org/10.1371/journal.pone.0209005.g007
https://doi.org/10.1371/journal.pone.0209005.g008
https://doi.org/10.1371/journal.pone.0209005
possible to detect RVA in a variety of animal species using NSP3 gene [49]. The sensitivity of
RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals
and in this context, the present study proposed an interesting study metrics using virus spread-
ing in the wild animals which inhabit forest fragments to indicate human population interven-
tions, with the goal of preventing the virus outbreaks leveraged on the unique geographic
characteristics of Brazil and its large number of species in Amazon.
Currently, no data have been described in the literature regarding the RVA detection using
real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan
et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR
and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that
showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle
(14.6%).
The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught
in Vihiga, Kenya [51]. Afterwards, several strains of RVA were detected by different molecular
techniques involving chiroptera, in several countries, including Kenya (E. helvum), China
(Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon
(E. helvum) and Brazil [31, 51–55]. The present study shows the occurrence of RVA in 17.98%
of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus
(28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%),
Diaemus iyoug and Glossophagine (3.12%).
Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous spe-
cies of the Phyllostomidae family, found throughout South America, Central America and
Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of
Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or
large, facilitating the dissemination of viral spores among the community within the habitat, as
observed in the present study. These findings show the importance of epidemiological data on
the studied species due to the lack of studies involving species of neotropical chiroptera,and it
is not possible to establish comparative parameters for these animals.
Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%,
respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG
that infect birds [57–58] and RVD in migratory birds [59], all were detected by RT-PCR assays
differently from the present study which detected the RVA by RT-qPCR involving a variety of
animal species.
On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably
because there are few animals of these species in the region, as well as few creations.
The study detected the presence of RVA in different species of animals both in areas near
the home and in areas located in fragments of forest, characterized as forest remnants, since
they were located in cities that suffered high environmental impacts due to vegetal extracti-
vism, pasture formation for cattle breeding, exploitation of natural resources, and direct
reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dis-
persion of RVA among communities of coexisting animals.
It is worth emphasizing that these animals have a greater contact with the human popula-
tions of the studied areas since they cohabit with the humans in the region, besides having a
high flow of movement between the forest extracts and environments chosen for the present
study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu
were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA,
but all were negative. It is notorious yet, the existence of different levels of degradation in the
studied environments, considering the presented data. The fragmentation of the forest gener-
ates many consequences on the Amazonian biota, being able to alter the diversity and the
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 12 / 18
https://doi.org/10.1371/journal.pone.0209005
composition of the animal communities in the fragments and even to interfere in the ecologi-
cal processes, without considering that the fragments of forest in the Amazon are influenced
by the climate, possibly facilitating the dispersion of pathogens by the environment, since the
wild animals detected in the present study are asymptomatic and have low viral load for RVA.
The occurrence of RVA in this population of animals may explain the possibility of disper-
sion of viral strains, since there is a proximity to the human population, besides the biological
characteristics of these species that may represent important sources for gastroenteric viruses,
along with the fact that all animals were asymptomatic for diarrhea.
Wild birds have unlimited flight capacity, were captured in an interface region between the
peridomicile and forest fragments and it is believed that this region has not been influenced by
anthropic activities such as those observed in the area of the present study. On the other hand,
the breeding method for poultry and canines close to homes and the forest ecosystem, as they
are created in the communities surveyed, probably facilitates direct contact with possible
sources of contamination, since in the areas the use of septic tanks is deficient and sometimes
non-existent, which may facilitate or even increase the risk of viral dispersion throughout the
environment.
The high rates of increase and the analysis of land use in the researched areas may be
important indicators of how these animals interact, since with deforestation, the populations
of wild animals seek refuge in nearby communities facilitating the dispersal of infectious
agents and the possible occurrence of carrier animals by direct contact or contamination of
the local environment.
To our knowledge, this is the first study in which a real-time PCR assay was applied for the
detection of RVA involving a wide variety of domestic and wild animals, facilitating practical
utility in epidemiological and molecular studies and assisting in a perspective in the elabora-
tion of sanitary control and monitoring, preventing possible outbreaks in the studied commu-
nities. The detection of positive animals was useful to monitor the infection of the agent in the
animal population and to provide an early warning signal to predict an impending epidemic
and a favorable risk for the human population, given the evidence of RVA circulation in the
different forest fragments.
In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of
RV in possible coexisting mixed infections clinically indistinguishable such as those caused by
other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus,
calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61].
Diarrhea associated with RVA infections in pigs is an important cause of increased mortal-
ity and economic losses in Europe. The most prevalent genotypes isolated from feces of Bel-
gian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of
combinations of genotypes G / P including; G3P[6], G4P[6], G5P[6], G4P[7], G5P[7], G9P[7],
G9P[13] and G9P[23]. On the other hand, in the present study it was possible to detect only P
[6] genotype, since majority of samples was asymptomatic for diarrhea.
Finding shows that different P genotypes of RVA strains interact with distinct blood group
histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into
the regional prevalence and increased zoonotic potential of some RVA of origin swine [63].
The genotype P[6] was identified in piglets in Brazil [64] and in Italy and Japan resembling
genotype P[6] human [65, 66].
In the population of animals studied the zoonotic transmission can be frequent, since the
animals live in contact with humans and in precarious sanitary conditions. In Brazil, this geno-
type was described in animal and human populations in studies of Luchs et al. [32]; Honma
et al. [67]; Araújo et al.[68]; Mascarenhas et al.[69] and Lorenzetti et al. [70] such studies
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 13 / 18
https://doi.org/10.1371/journal.pone.0209005
corroborate the importance of continuing to monitor genotypes to verify if uncommon strains
or new strains are emerging and can infect animal populations or inter-species transmissions.
Regarding the genotype P [4], itwas most detected in our samples in bats, dogs, swine and
feline. This genotype is not common in animals, being more detected in human and environ-
mental samples in various parts of the world and included our region [71]. It is important to
emphasize that the indicators of environmental contamination in Brazil are significant and
contribute to the possibility of human-animal transmission [71]. Such data need further inves-
tigation in later work to better characterize the interspecies transmission, since the occurrence
of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed
population around and points to the potential risk of infection by the possible exposure of
individuals susceptible. Our findings may be useful for tracking fecal contamination in the
environment using animals as possible sources thus minimizing the risk of infection by expo-
sure to susceptible individuals, in this case different animal species or even human
populations.
Conclusions and Significance
RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed sam-
ples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is
necessary to conduct strategies for the monitoring and control of the animals in the areas stud-
ied in the human population as well as in other speciesof animals, as well as the implementa-
tion of preventive measures aimed at future outbreaks in communities animals in the resident
population in these impacted areas. Therefore, the present study is unprecedented in the
region and in the country in relation to the research of RVA in wild animals. It is noteworthy
that, although the quality of the analyzed samples is characterized as low detectable viral load,
the technique presented a good analytical response in the detection of the source animals for
RVA, facilitating the selection of the samples for future genetic characterization tests.
Acknowledgments
The authors would like to thank the invaluable technical support provided by Euzenir
Menezes, Livia Casseb and Luana Soares. They are also grateful to the Laboratory of Geopro-
cessing (LABGEO), and the staff of the Library, both of the Evandro Chagas Institute. They
also thank the health workers and community leaders of the cities surveyed. Comments on the
manuscript of Dr. Ana Cecı́lia Ribeiro Cruz were acknowledged with gratitude. Finally, we
thank CAPES for the granting of doctoral and postdoctoral scholarships. BARROS BCV is the
beneficiary of the Capes concession; CHAGAS EHN and MASCARENHAS JDP are beneficia-
ries of grants from CNPq.
Author Contributions
Data curation: Ricardo José de Paula Souza e Guimarães.
Funding acquisition: Joana D’Arc Pereira Mascarenhas.
Investigation: Jose Wandilson Barboza Duarte Júnior, Diego Pereira, Edvaldo Tavares da
Penha Junior, Julia Rezende Silva.
Methodology: Bruno de Cássio Veloso de Barros, Elaine Nunes Chagas, Laila Graziela Ribeiro,
Delana Andreza Melo Bezerra, Helder Henrique Costa Pinheiro, Sylvia de Fátima dos San-
tos Guerra, Ricardo José de Paula Souza e Guimarães.
Project administration: Bruno de Cássio Veloso de Barros, Joana D’Arc Pereira Mascarenhas.
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 14 / 18
https://doi.org/10.1371/journal.pone.0209005
Supervision: Joana D’Arc Pereira Mascarenhas.
Validation: Renato Silva Bandeira.
Writing – original draft: Bruno de Cássio Veloso de Barros, Luna Wanessa Bezerra.
Writing – review & editing: Renato Silva Bandeira, Sylvia de Fátima dos Santos Guerra, Joana
D’Arc Pereira Mascarenhas.
References
1. Schatzmayr H (2001) Viroses emergentes e reemergentes. Cadernos de Saúde Pública 17: S209–
S213. https://doi.org/10.1590/s0102-311x2001000700031
2. Vasconcelos P (2010) Yellow fever in Brazil: thoughts and hypotheses on the emergence in previously
free areas. Revista de Saúde Pública 44: 1144–1149. https://doi.org/10.1590/s0034-
89102010005000046 PMID: 21109907
3. Zanella J (2016) Zoonoses emergentes e reemergentes e sua importância para saúde e produção animal.
Pesquisa Agropecuária Brasileira 51: 510–519. https://doi.org/10.1590/s0100-204x2016000500011
4. Dulgheroff A, Silva G, Naveca F, Oliveira A, Domingues A (2016) Diversity of group A rotavirus genes
detected in the Triângulo Mineiro region, Minas Gerais, Brazil. Brazilian Journal of Microbiology 47:
731–740. https://doi.org/10.1016/j.bjm.2016.04.012 PMID: 27266629
5. UNICEF (2018) UNICEF Brasil—Imprensa—UNICEF e OMS lançam relatório sobre diarreia, a
segunda maior causa de mortalidade infantil. Uniceforg. Available: https://www.unicef.org/brazil/pt/
media_16165.html. Accessed 27 October 2017.
6. Ito H, Sugiyama M, Masubuchi K, Mori Y, Minamoto N (2001) Complete nucleotide sequence of a group
A avian rotavirus genome and a comparison with its counterparts of mammalian rotaviruses. Virus
Research 75: 123–138. https://doi.org/10.1016/s0168-1702(01)00234-9 PMID: 11325467
7. Rotavirus Classification Working Group: RCWG (2018). Regakuleuvenbe. Available: https://rega.
kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg. Accessed 25 February 2018.
8. Dhama K, Chauhan R, Mahendran M, Malik S (2008) Rotavirus diarrhea in bovines and other domestic
animals. Veterinary Research Communications 33: 1–23. https://doi.org/10.1007/s11259-008-9070-x
PMID: 18622713
9. De Grazia S, Martella V, Giammanco G, Gòmara M, Ramirez S, Cascio A et al. (2007) Canine-Origin
G3P[3] Rotavirus Strain in Child with Acute Gastroenteritis. Emerging Infectious Diseases 13: 1091–
1093. https://doi.org/10.3201/eid1307.070239 PMID: 18214189
10. Luchs A, Cilli A, Morillo S, Carmona R, Timenetsky M (2012) Rare G3P[3] rotavirus strain detected in
Brazil: Possible human–canine interspecies transmission. Journal of Clinical Virology 54: 89–92.
https://doi.org/10.1016/j.jcv.2012.01.025 PMID: 22398035
11. Doan Y, Nakagomi T, Aboudy Y, Silberstein I, Behar-Novat E, Nakagomi O et al. (2012) Identification
by Full-Genome Analysis of a Bovine Rotavirus Transmitted Directly to and Causing Diarrhea in a
Human Child. Journal of Clinical Microbiology 51: 182–189. https://doi.org/10.1128/JCM.02062-12
PMID: 23115264
12. Papp H, László B, Jakab F, Ganesh B, De Grazia S, Matthijnssens J et al. (2013) Review of group A
rotavirus strains reported in swine and cattle. Veterinary Microbiology 165: 190–199. https://doi.org/10.
1016/j.vetmic.2013.03.020 PMID: 23642647
13. Mihalov-Kovács E, Gellért Á, Marton S, Farkas S, Fehér E, Oldal M et al. (2015) Candidate New Rotavi-
rus Species in Sheltered Dogs, Hungary. Emerging Infectious Diseases 21: 660–663. https://doi.org/
10.3201/eid2104.141370 PMID: 25811414
14. Bányai K, Kemenesi G, Budinski I, Földes F, Zana B, Marton S et al. (2017) Candidate new rotavirus
species in Schreiber’s bats, Serbia. Infection, Genetics and Evolution 48: 19–26. https://doi.org/10.
1016/j.meegid.2016.12.002 PMID: 27932285
15. Matthijnssens J, Otto P, Ciarlet M, Desselberger U, Van Ranst M, Johne R (2012) VP6-sequence-
based cutoff values as a criterion for rotavirus species demarcation. Archives of Virology 157: 1177–
1182. https://doi.org/10.1007/s00705-012-1273-3 PMID: 22430951
16. ESTES M., GREENBERG H. Rotaviruses. In: FIELDS B.N., Knipe D.M., Howley P.M. (Eds.). Fields
Virology, 6th ed. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia, 2013. cap 45 p.
1347–1401.
17. Ciarlet M, Liprandi F, Conner M, Estes M (2000) Species specificity and interspecies relatedness of
NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses. Archives of Virol-
ogy 145: 371–383. https://doi.org/10.1007/s007050050029 PMID: 10752559
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 15 / 18
https://doi.org/10.1590/s0102-311x2001000700031
https://doi.org/10.1590/s0034-89102010005000046
https://doi.org/10.1590/s0034-89102010005000046
http://www.ncbi.nlm.nih.gov/pubmed/21109907
https://doi.org/10.1590/s0100-204x2016000500011
https://doi.org/10.1016/j.bjm.2016.04.012
http://www.ncbi.nlm.nih.gov/pubmed/27266629
https://www.unicef.org/brazil/pt/media_16165.html
https://www.unicef.org/brazil/pt/media_16165.html
https://doi.org/10.1016/s0168-1702(01)00234-9
http://www.ncbi.nlm.nih.gov/pubmed/11325467
https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg
https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg
https://doi.org/10.1007/s11259-008-9070-x
http://www.ncbi.nlm.nih.gov/pubmed/18622713
https://doi.org/10.3201/eid1307.070239
http://www.ncbi.nlm.nih.gov/pubmed/18214189
https://doi.org/10.1016/j.jcv.2012.01.025
http://www.ncbi.nlm.nih.gov/pubmed/22398035
https://doi.org/10.1128/JCM.02062-12
http://www.ncbi.nlm.nih.gov/pubmed/23115264
https://doi.org/10.1016/j.vetmic.2013.03.020
https://doi.org/10.1016/j.vetmic.2013.03.020
http://www.ncbi.nlm.nih.gov/pubmed/23642647
https://doi.org/10.3201/eid2104.141370
https://doi.org/10.3201/eid2104.141370
http://www.ncbi.nlm.nih.gov/pubmed/25811414
https://doi.org/10.1016/j.meegid.2016.12.002
https://doi.org/10.1016/j.meegid.2016.12.002
http://www.ncbi.nlm.nih.gov/pubmed/27932285
https://doi.org/10.1007/s00705-012-1273-3
http://www.ncbi.nlm.nih.gov/pubmed/22430951https://doi.org/10.1007/s007050050029
http://www.ncbi.nlm.nih.gov/pubmed/10752559
https://doi.org/10.1371/journal.pone.0209005
18. Clement C (1999) 1492 and the loss of amazonian crop genetic resources. I. The relation between
domestication and human population decline. Economic Botany 53: 188–202. https://doi.org/10.1007/
bf02866498
19. Daszak P (2000) Emerging Infectious Diseases of Wildlife—Threats to Biodiversity and Human Health.
Science 287: 443–449. https://doi.org/10.1126/science.287.5452.443 PMID: 10642539
20. Hayman D, Bowen R, Cryan P, McCracken G, O’Shea T, Peel A J et al. (2012) Ecology of Zoonotic
Infectious Diseases in Bats: Current Knowledge and Future Directions. Zoonoses and Public Health
60: 2–21. https://doi.org/10.1111/zph.12000 PMID: 22958281
21. Cruz Junior E, Salvarani F, Silva R, Silva M, Lobato F, Guedes RMC (2013) A surveillance of entero-
pathogens in piglets from birth to seven days of age in Brazil. Pesquisa Veterinária Brasileira 33: 963–
969. https://doi.org/10.1590/s0100-736x2013000800002
22. Anyamba A, Linthicum K, Small J, Collins K, Tucker C, Pak EW et al. (2012) Climate Teleconnections
and Recent Patterns of Human and Animal Disease Outbreaks. PLoS Neglected Tropical Diseases 6:
e1465. https://doi.org/10.1371/journal.pntd.0001465 PMID: 22292093
23. Vlasova A, Amimo J, Saif L (2017) Porcine Rotaviruses: Epidemiology, Immune Responses and Con-
trol Strategies. Viruses 9: 48. https://doi.org/10.3390/v9030048 PMID: 28335454
24. Jing J, Søegaard K, Cong W, Eriksen J (2017) Species Diversity Effects on Productivity, Persistence
and Quality of Multispecies Swards in a Four-Year Experiment. PLOS ONE 12: e0169208. https://doi.
org/10.1371/journal.pone.0169208 PMID: 28046004
25. Okadera K, Abe M, Ito N, Morikawa S, Yamasaki A, Masatani T et al. (2013) Evidence of natural trans-
mission of group A rotavirus between domestic pigs and wild boars (Sus scrofa) in Japan. Infection,
Genetics and Evolution 20: 54–60. https://doi.org/10.1016/j.meegid.2013.07.029 PMID: 23958658
26. Otto P, Rosenhain S, Elschner M, Hotzel H, Machnowska P, Trojnar E et al. (2015) Detection of rotavi-
rus species A, B and C in domestic mammalian animals with diarrhoea and genotyping of bovine spe-
cies A rotavirus strains. Veterinary Microbiology 179: 168–176. https://doi.org/10.1016/j.vetmic.2015.
07.021 PMID: 26223422
27. Pauly M, Oni O, Sausy A, Owoade A, Adeyefa C, Muller CP et al. (2017) Molecular epidemiology of
Avian Rotaviruses Group A and D shed by different bird species in Nigeria. Virology Journal 14. https://
doi.org/10.1186/s12985-017-0778-5 PMID: 28606119
28. Trojnar E, Reetz J, Twardziok S, Sachsenröder J, Otto PH, Johne R et al. (2013) Identification of an
avian group A rotavirus containing a novel VP4 gene with a close relationship to those of mammalian
rotaviruses. Journal of General Virology 94: 136–142. https://doi.org/10.1099/vir.0.047381-0 PMID:
23052396
29. Malik Y, Sharma K, Vaid N, Chakravarti S, Chandrashekar K, Basera SS et al. (2012) Frequency of
group A rotavirus with mixed G and P genotypes in bovines: predominance of G3 genotype and its
emergence in combination with G8/G10 types. Journal of Veterinary Science 13: 271. https://doi.org/
10.4142/jvs.2012.13.3.271 PMID: 23006956
30. Hassine-Zaafrane M, Ben Salem I, Sdiri-Loulizi K, Kaplon J, Bouslama L, Aouni Z et al. (2014) Distribu-
tion of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diar-
rhoea. Journal of Applied Microbiology 116: 1387–1395. https://doi.org/10.1111/jam.12469 PMID:
24905214
31. Asano K, Gregori F, Hora A, Scheffer K, Fahl W, Iamamoto K et al. (2016) Group A rotavirus in Brazilian
bats: description of novel T15 and H15 genotypes. Archives of Virology 161: 3225–3230. https://doi.
org/10.1007/s00705-016-3010-9 PMID: 27518402
32. Luchs A, Timenetsky M (2016) Group A rotavirus gastroenteritis: post-vaccine era, genotypes and zoo-
notic transmission. Einstein (São Paulo) 14: 278–287. https://doi.org/10.1590/s1679-45082016rb3582
PMID: 27462899
33. Silva F, Gregori F, Gonçalves A, Samara S, Buzinaro M (2012) Molecular characterization of group A
bovine rotavirus in southeastern and central-western Brazil, 2009–2010. Pesquisa Veterinária Brasi-
leira 32: 237–242. https://doi.org/10.1590/s0100-736x2012000300010
34. Medeiros T, Lorenzetti E, Alfieri A, Alfieri A (2014) Severe diarrhea outbreak in beef calves (Bos indicus)
caused by G6P[11], an emergent genotype of bovine rotavirus group A. Pesquisa Veterinária Brasileira
34: 717–722. https://doi.org/10.1590/s0100-736x2014000800001
35. INPE—Instituto Nacional de Pesquisas Espaciais (2018). Inpebr. Available: http://www.inpe.br.
Accessed 15 January 2018.
36. Plowright R, Eby P, Hudson P, Smith I, Westcott D, Bryden WL et al. (2014) Ecological dynamics of
emerging bat virus spillover. Proceedings of the Royal Society B: Biological Sciences 282: 20142124–
20142124. https://doi.org/10.1098/rspb.2014.2124 PMID: 25392474
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 16 / 18
https://doi.org/10.1007/bf02866498
https://doi.org/10.1007/bf02866498
https://doi.org/10.1126/science.287.5452.443
http://www.ncbi.nlm.nih.gov/pubmed/10642539
https://doi.org/10.1111/zph.12000
http://www.ncbi.nlm.nih.gov/pubmed/22958281
https://doi.org/10.1590/s0100-736x2013000800002
https://doi.org/10.1371/journal.pntd.0001465
http://www.ncbi.nlm.nih.gov/pubmed/22292093
https://doi.org/10.3390/v9030048
http://www.ncbi.nlm.nih.gov/pubmed/28335454
https://doi.org/10.1371/journal.pone.0169208
https://doi.org/10.1371/journal.pone.0169208
http://www.ncbi.nlm.nih.gov/pubmed/28046004
https://doi.org/10.1016/j.meegid.2013.07.029
http://www.ncbi.nlm.nih.gov/pubmed/23958658
https://doi.org/10.1016/j.vetmic.2015.07.021
https://doi.org/10.1016/j.vetmic.2015.07.021
http://www.ncbi.nlm.nih.gov/pubmed/26223422
https://doi.org/10.1186/s12985-017-0778-5
https://doi.org/10.1186/s12985-017-0778-5
http://www.ncbi.nlm.nih.gov/pubmed/28606119
https://doi.org/10.1099/vir.0.047381-0
http://www.ncbi.nlm.nih.gov/pubmed/23052396
https://doi.org/10.4142/jvs.2012.13.3.271
https://doi.org/10.4142/jvs.2012.13.3.271
http://www.ncbi.nlm.nih.gov/pubmed/23006956
https://doi.org/10.1111/jam.12469
http://www.ncbi.nlm.nih.gov/pubmed/24905214
https://doi.org/10.1007/s00705-016-3010-9
https://doi.org/10.1007/s00705-016-3010-9
http://www.ncbi.nlm.nih.gov/pubmed/27518402
https://doi.org/10.1590/s1679-45082016rb3582
http://www.ncbi.nlm.nih.gov/pubmed/27462899
https://doi.org/10.1590/s0100-736x2012000300010
https://doi.org/10.1590/s0100-736x2014000800001
http://www.inpe.br
https://doi.org/10.1098/rspb.2014.2124
http://www.ncbi.nlm.nih.gov/pubmed/25392474
https://doi.org/10.1371/journal.pone.0209005
37. Kattoor J, Saurabh S, Malik Y, Sircar S, Dhama K, Ghosh S et al. (2017) Unexpected detection of por-
cine rotavirus C strains carrying human origin VP6 gene. Veterinary Quarterly 37: 252–261. https://doi.
org/10.1080/01652176.2017.1346849 PMID: 28643555
38. Dóró R, Farkas S, Martella V, Bányai K (2015) Zoonotic transmission of rotavirus: surveillance and con-
trol. Expert Review of Anti-infective Therapy 13: 1337–1350. https://doi.org/10.1586/14787210.2015.
1089171 PMID: 26428261
39. Zhou X, Wang Y, Ghosh S, Tang W, Pang B, Liu MQ et al. (2015) Genomic characterization of G3P[6],
G4P[6] and G4P[8] human rotaviruses from Wuhan, China: Evidence for interspecies transmission and
reassortment events. Infection, Genetics and Evolution 33: 55–71. https://doi.org/10.1016/j.meegid.
2015.04.010 PMID: 25891280
40. Zeng S, Halkosalo A, Salminen M, Szakal E, Puustinen L, Vesikari T (2008) One-step quantitative RT-
PCR for the detection of rotavirus in acute gastroenteritis. Journal of Virological Methods 153: 238–
240. https://doi.org/10.1016/j.jviromet.2008.08.004 PMID: 18765254
41. Mijatovic-Rustempasic S, Esona M, Williams A, Bowen M (2016) Sensitive and specificnested PCR
assay for detection of rotavirus A in samples with a low viral load. Journal of Virological Methods 236:
41–46. https://doi.org/10.1016/j.jviromet.2016.07.007 PMID: 27421626
42. Alvares C, Stape J, Sentelhas P, de Moraes Gonçalves J, Sparovek G (2013) Köppen’s climate classifi-
cation map for Brazil. Meteorologische Zeitschrift 22: 711–728. https://doi.org/10.1127/0941-2948/
2013/0507
43. Brasil I (2018) PRODES—Coordenação-Geral de Observação da Terra. Obtinpebr. Available: http://
www.obt.inpe.br/OBT/assuntos/programas/amazonia/prodes. Accessed 5 February 2018.
44. Almeida C, Coutinho A, Esquerdo J, Adami M, Venturieri A, Diniz CD et al. (2016) High spatial resolu-
tion land use and land cover mapping of the Brazilian Legal Amazon in 2008 using Landsat-5/TM and
MODIS data. Acta Amazonica 46: 291–302. https://doi.org/10.1590/1809-4392201505504
45. Gomes M, Monteiro A, Nogueira Filho V, Gonçalves C (2018) Áreas propı́cias para o ataque de morce-
gos hematófagos Desmodus rotundus em bovinos na região de São João da Boa Vista, Estado de São
Paulo.
46. Colwell R (2018) EstimateS: Biodiversity Estimation. Viceroycoloradoedu.Available:http://viceroy.
colorado.edu/estimates/. Accessed 24 January 2018.
47. Brower J, Zar J, Ende C (1998) Field and laboratory methods for general ecology. Boston, Mass.:
WCB McGraw-Hill.
48. INPE—Instituto Nacional de Pesquisas Espaciais (2018). Inpebr. Available: http://www.inpe.br.
Accessed 15 January 2018.
49. Espy M, Uhl J, Sloan L, Buckwalter S, Jones M, Vetter EA et al. (2006) Real-Time PCR in Clinical Micro-
biology: Applications for Routine Laboratory Testing. Clinical Microbiology Reviews 19: 595–595.
https://doi.org/10.1128/cmr.00022-06
50. Soltan M, Tsai Y, Lee P, Tsai C, Chang H, Wang HT et al. (2016) Comparison of electron microscopy,
ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A
(RVA) in feces of different animal species. Journal of Virological Methods 235: 99–104. https://doi.org/
10.1016/j.jviromet.2016.05.006 PMID: 27180038
51. Esona M, Mijatovic-Rustempasic S, Conrardy C, Tong S, Kuzmin I, Agwanda B et al. (2010) Reassor-
tant Group A Rotavirus from Straw-colored Fruit Bat (Eidolon helvum). Emerging Infectious Diseases
16: 1844–1852. https://doi.org/10.3201/eid1612.101089 PMID: 21122212
52. Dacheux L, Cervantes-Gonzalez M, Guigon G, Thiberge J, Vandenbogaert M, Maufrais C et al. (2014)
A Preliminary Study of Viral Metagenomics of French Bat Species in Contact with Humans: Identifica-
tion of New Mammalian Viruses. PLoS ONE 9: e87194. https://doi.org/10.1371/journal.pone.0087194
PMID: 24489870
53. He B, Yang F, Yang W, Zhang Y, Feng Y, Zhou J et al. (2013) Characterization of a Novel G3P[3] Rota-
virus Isolated from a Lesser Horseshoe Bat: a Distant Relative of Feline/Canine Rotaviruses. Journal of
Virology 87: 12357–12366. https://doi.org/10.1128/JVI.02013-13 PMID: 24027312
54. Xia L, Fan Q, He B, Xu L, Zhang F, Hu T et al. (2014) The complete genome sequence of a G3P[10]
Chinese bat rotavirus suggests multiple bat rotavirus inter-host species transmission events. Infection,
Genetics and Evolution 28: 1–4. https://doi.org/10.1016/j.meegid.2014.09.005 PMID: 25218875
55. Yinda C, Zell R, Deboutte W, Zeller M, Conceição-Neto N, Heylen E et al. (2017) Highly diverse popula-
tion of Picornaviridae and other members of the Picornavirales, in Cameroonian fruit bats. BMC Geno-
mics 18. https://doi.org/10.1186/s12864-017-3632-7 PMID: 28335731
56. Barquez R, Perez S, Miller B, Diaz M (2018) Desmodus rotundus (Common Vampire Bat, Vampire
Bat). Iucnredlistorg. Available: http://www.iucnredlist.org/details/6510/0. Accessed 13 January 2018.
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 17 / 18
https://doi.org/10.1080/01652176.2017.1346849
https://doi.org/10.1080/01652176.2017.1346849
http://www.ncbi.nlm.nih.gov/pubmed/28643555
https://doi.org/10.1586/14787210.2015.1089171
https://doi.org/10.1586/14787210.2015.1089171
http://www.ncbi.nlm.nih.gov/pubmed/26428261
https://doi.org/10.1016/j.meegid.2015.04.010
https://doi.org/10.1016/j.meegid.2015.04.010
http://www.ncbi.nlm.nih.gov/pubmed/25891280
https://doi.org/10.1016/j.jviromet.2008.08.004
http://www.ncbi.nlm.nih.gov/pubmed/18765254
https://doi.org/10.1016/j.jviromet.2016.07.007
http://www.ncbi.nlm.nih.gov/pubmed/27421626
https://doi.org/10.1127/0941-2948/2013/0507
https://doi.org/10.1127/0941-2948/2013/0507
http://www.obt.inpe.br/OBT/assuntos/programas/amazonia/prodes
http://www.obt.inpe.br/OBT/assuntos/programas/amazonia/prodes
https://doi.org/10.1590/1809-4392201505504
http://viceroy.colorado.edu/estimates/
http://viceroy.colorado.edu/estimates/
http://www.inpe.br
https://doi.org/10.1128/cmr.00022-06
https://doi.org/10.1016/j.jviromet.2016.05.006
https://doi.org/10.1016/j.jviromet.2016.05.006
http://www.ncbi.nlm.nih.gov/pubmed/27180038
https://doi.org/10.3201/eid1612.101089
http://www.ncbi.nlm.nih.gov/pubmed/21122212
https://doi.org/10.1371/journal.pone.0087194
http://www.ncbi.nlm.nih.gov/pubmed/24489870
https://doi.org/10.1128/JVI.02013-13
http://www.ncbi.nlm.nih.gov/pubmed/24027312
https://doi.org/10.1016/j.meegid.2014.09.005
http://www.ncbi.nlm.nih.gov/pubmed/25218875
https://doi.org/10.1186/s12864-017-3632-7
http://www.ncbi.nlm.nih.gov/pubmed/28335731
http://www.iucnredlist.org/details/6510/0
https://doi.org/10.1371/journal.pone.0209005
57. Bezerra D, da Silva R, Kaiano J, de Souza Oliveira D, Gabbay Y, Linhares AC et al. (2014) Detection,
epidemiology and characterization of VP6 and VP7 genes of group D rotavirus in broiler chickens.
Avian Pathology 43: 238–243. https://doi.org/10.1080/03079457.2014.913097 PMID: 24875189
58. Mascarenhas J, Bezerra D, Silva R, Silva M, Júnior E, Soares LS (2016) Detection of the VP6 gene of
group F and G rotaviruses in broiler chicken fecal samples from the Amazon region of Brazil. Archives
of Virology 161: 2263–2268. https://doi.org/10.1007/s00705-016-2882-z PMID: 27154557
59. Guerreiro A, Moraes C, Marinho A, Barros B, Bezerra D, Bandeira RS et al. (2018) Investigation of
Enteric Viruses in the Feces of Neotropical Migratory Birds Captured on the Coast of the State of Pará,
Brazil. Revista Brasileira de Ciência Avı́cola 20: 161–168. https://doi.org/10.1590/1806-9061-2017-
0589
60. Jing Z, Zhang X, Shi H, Chen J, Shi D, Dong H et al. (2017) A G3P[13] porcine group A rotavirus emerg-
ing in China is a reassortant and a natural recombinant in the VP4 gene. Transboundary and Emerging
Diseases 65: e317–e328. https://doi.org/10.1111/tbed.12756 PMID: 29148270
61. Waruhiu C, Ommeh S, Obanda V, Agwanda B, Gakuya F, Ge XY et al. (2017) Molecular detection of
viruses in Kenyan bats and discovery of novel astroviruses, caliciviruses and rotaviruses. Virologica
Sinica 32: 101–114. https://doi.org/10.1007/s12250-016-3930-2 PMID: 28393313
62. Theuns S,Desmarets LM.,Heylen E, Zeller M, Dedeurwaerder A, Roukaerts ID et al. (2014) Porcine
group A rotaviruses with heterogeneous VP7 and VP4 genotype combinations can be found together
with enteric bacteria on Belgian swine farms. Vet. Microbiol. 172, 23–34. https://doi.org/10.1016/j.
vetmic.2014.04.002 PMID: 24837191
63. Liu Y, Huang P, Tan M, Liu Y, Biesiada J, Meller J et al. (2012) Rotavirus VP8*: phylogeny, host range,
and interaction with histo-blood group antigens. J. Virol., 86, 9899–9910, https://doi.org/10.1128/JVI.
00979-12 PMID: 22761376
64. Tonietti PO, Hora AS, Silva FD, Ruiz VL, Gregori F. (2013) Phylogenetic analysis of the VP4 and VP7
genes of group A rotavirus in the State of São Paulo, Brazil: first identification of G5P [23] in piglets. J
Clin Microbiol.; 51 (8): 2750–3, https://doi.org/10.1128/JCM.01175-13 PMID: 23761160
65. Martella V, Bányai K, Ciarlet M., Iturriza-Gómara M, Lorusso E, De Grazia S et al. (2006) Relationships
between rotaviruses P6 porcine and human: evidence that the different human strains of [6]originated
from multiple interspecies transmission events. Virology 344: 509–519, https://doi.org/10.1016/j.virol.
2005.08.029 PMID: 16194556
66. Teodoroff TA, Tsunemitsu H, Okamoto K, Katsuda K, Kohmoto M, Kawashima K et al. (2005). Preva-
lence of G9 porcine rotavirus in Japanese piglets with diarrhea: close relationship of their VP7 genes to
those of recent humans. Strains G9. J. Clin. Microbiol. 43: 1377–1384, https://doi.org/10.1128/JCM.43.
3.1377-1384.2005 PMID: 15750112
67. Honma S, Chizhikov V, Santos N, Tatsumi M, Timenetsky MC, Linhares AC et al. (2007) Development
and validation of DNA microarray for genotyping group A rotavirus VP4 (P [4], P [6], P [8], P [9], and P
[14]) and VP7 (G1 to G6, G8 to G10, and G12) genes. Journal of clinical microbiology, 45: 2641–2648,
https://doi.org/10.1128/JCM.00736-07 PMID: 17567783
68. Araújo I, Assis RM, Fialho AM, Mascarenhas JD, Heinemann MB, Leite JP.(2007) Brazilian P [8], G1, P
[8], G5, P [8], G9, and P [4], G2 rotavirus strains: nucleotide sequence and phylogenetic analysis.Jour-
nal of medical virology; 79: 995–1001, https://doi.org/10.1002/jmv.20918 PMID: 17516538
69. Mascarenhas JDP, Linhares AC, Gabbay YB, Lima CS, Guerra SFS, Soares LS. et al. (2007) Molecular
characterization of VP4 and NSP4 genes from rotavirus strains infecting neonates and young children
in Belem, Brazil. Virus research; 126: 149–158, https://doi.org/10.1016/j.virusres.2007.02.010 PMID:
17376554
70. Lorenzetti E, Medeiros TNS, Alfieri AF, Alfieri AA (2011). Genetic heterogeneity of wild-type G4P [6]
porcine rotavirus strains detected in a diarrhea outbreak in a regularly vaccinated pig herd. Veterinary
microbiology; 154: 191–196, https://doi.org/10.1016/j.vetmic.2011.06.026 PMID: 21775076
71. Fumian TM, Victoria M, Vieira CB, Fioretti JM, Rocha MS, Prado T et al. (2018) Enteric viruses’ dissemi-
nation in a private reserve of natural heritage. Letters in applied microbiology; 66: 313–320, 2018.
https://doi.org/10.1111/lam.12848 PMID: 29330866
Rotavirus A in wild and domestic animals
PLOS ONE | https://doi.org/10.1371/journal.pone.0209005 December 18, 2018 18 / 18
https://doi.org/10.1080/03079457.2014.913097
http://www.ncbi.nlm.nih.gov/pubmed/24875189
https://doi.org/10.1007/s00705-016-2882-z
http://www.ncbi.nlm.nih.gov/pubmed/27154557
https://doi.org/10.1590/1806-9061-2017-0589
https://doi.org/10.1590/1806-9061-2017-0589
https://doi.org/10.1111/tbed.12756
http://www.ncbi.nlm.nih.gov/pubmed/29148270
https://doi.org/10.1007/s12250-016-3930-2
http://www.ncbi.nlm.nih.gov/pubmed/28393313
https://doi.org/10.1016/j.vetmic.2014.04.002
https://doi.org/10.1016/j.vetmic.2014.04.002
http://www.ncbi.nlm.nih.gov/pubmed/24837191
https://doi.org/10.1128/JVI.00979-12
https://doi.org/10.1128/JVI.00979-12
http://www.ncbi.nlm.nih.gov/pubmed/22761376
https://doi.org/10.1128/JCM.01175-13
http://www.ncbi.nlm.nih.gov/pubmed/23761160
https://doi.org/10.1016/j.virol.2005.08.029
https://doi.org/10.1016/j.virol.2005.08.029
http://www.ncbi.nlm.nih.gov/pubmed/16194556
https://doi.org/10.1128/JCM.43.3.1377-1384.2005
https://doi.org/10.1128/JCM.43.3.1377-1384.2005
http://www.ncbi.nlm.nih.gov/pubmed/15750112
https://doi.org/10.1128/JCM.00736-07
http://www.ncbi.nlm.nih.gov/pubmed/17567783
https://doi.org/10.1002/jmv.20918
http://www.ncbi.nlm.nih.gov/pubmed/17516538
https://doi.org/10.1016/j.virusres.2007.02.010
http://www.ncbi.nlm.nih.gov/pubmed/17376554
https://doi.org/10.1016/j.vetmic.2011.06.026
http://www.ncbi.nlm.nih.gov/pubmed/21775076
https://doi.org/10.1111/lam.12848
http://www.ncbi.nlm.nih.gov/pubmed/29330866
https://doi.org/10.1371/journal.pone.0209005